Current estimates indicate that 25-30% of translated proteins enter the secretory pathway In eukaryotic cells. After folding and assembly of nascent secretory proteins in the endoplasmic reticulum (ER), the coat protein complex II (COPII) sorts folded cargo into transport vesicles that bud from the ER and are targeted to cis- Golgi compartments. Genetic and proteomic approaches have identified many of the components required for efficient transport between the ER and Golgi complex. However, the mechanisms by which diverse secretory cargo are sorted into COPII vesicles and how budded vesicles are then targeted to Golgi acceptor membranes remain obscure. Our research program applies a multidisciplinary approach to define molecular mechanisms that catalyze receptor-dependent sorting of secretory cargo into COPII vesicles and vesicular transport to the Golgi complex using yeast as a model organism. We exploit a cell-free transport assay that proceeds through the biochemically resolvable stages of COPIi-dependent cargo selection and vesicle budding, Uso1p-dependent vesicle tethering, and SNARE protein-dependent membrane fusion. We have reproduced these stages with isolated membranes and purified soluble molecules. The long-term goal of my research program is to elucidate the catalytic mechanisms undertying these events though analysis of stage- specific assays and reconstitution experiments with defined protein and lipid fractions. Our recent progress has identified transmembrane cargo receptors (e.g. Erv26p and Erv29p) that select secretory proteins into COPII vesicles and function with the ER quality control pathway. In addition, we have made progress in characterizing specific lipid requirements, including PI(4)P, in distinct stages of ER to Golgi transport, in the next funding period we plan test molecular models to determine how cargo binding to export receptors is regulated; how cargo receptors function in ER quality control; the role of specific proteins and lipid species in organizing ER export sites and COPII budding; and the role of tethering factors and Pl(4) in transport to the Golgi complex. RELEVANCE (See instnjctions); These experimental aims are designed to address fundamental questions on how secretory proteins are selectively exported from the ER and directed to the Golgi complex. This intracellular transport step is essential for cell growth and regulation. Therefore these studies are basic for understanding numerous health related issues including cholesterol regulation, cystic fibrosis and diabetes.